Flowering hormone from carnauba wax and its preparation



United States Patent O 3,197,4s2 FLQWERKNG HGRMSNE FRGM CARNAUBA WAX AND lTS PREE ARATEON Ray H. Roberts and liurdean E. truclrrneyer, Madison,

Wis., assignors to Wisconsin Alumni Research Foundation, Idadison, Wis, a corporation of Wisconsin No Drawing. Filed Dec. 27, 1961, er. No. 162,575 Claims. (Cl. 260-2365) The present invention relates to oil-soluble hormone or hormone-like growth substances which can be used to assist blossoming in flowering plants. More specifically, the present invention relates to a process of obtaining auxin-free, including anti-auxin-free, antbogens" (Greek, for blossom generating) from anthogen-containing materials such as wax including carnauba wax, shellac wax and the like, and the resulting composition.

Our prior patent, No. 2,937,206 (May 17, 1960), describes a process of obtaining a blossom-inducing substance (florigen) from parts of flowering plants such as foliage and the like including carnauba wax obtained from the leaves of the flowering Brazilian wax palm. In our prior patent, supra, the blossom-inducing substance, Pram tion A, is identified as being antiauxin in character, i.e., it acts as a plant regulator in that its presence provides for normal anatomical and physiological development of tissue, as distinguished from auxin-like Fraction C, the presence of which makes for abnormal or excess tissue growth. Also, as pointed out in our prior patent, supra, while Fraction A, free from auxin-like Fraction C, can be employed for the partial induction of flowering, it appears that anti-auxin Fraction A belongs to the physiological complex associated with blossom initiation rather than the preceding period of flora induction.

In continued investigations it was discovered that three physiologically active anthogens are present in carnauba wax and like wax material and that the anthogens can be readily recovered, free from both auxin and anti-auxin material. One of the preferred procedures is as follows:

(1) Extract the commercial wax with acetone at room temperature. This step is optional but is a preferred means for removing unwanted auxin-like material when present in the wax.

(2) Extract the wax with a refined hydrocarbon solvent, e.g., Skelly C a distillate cut of crude oil which is predominantly n-heptane and which has a boiling point from S d-100 C. At room temperature (about 22 C.) anthogen I is extracted by this step and can be recovered by evaporation of the solvent. Anthogen I, the inducer, is associated with inducing flowering in plants.

(3) Extract the wax residue remaining from step (2) above, with chloroform. At room temperature anthogen H is extracted by this step and can be recovered by evaporation of the solvent. Anthogen II, the initiator, is associated with the initiation of flowering in plants.

(4) Dissolve the wax residue remaining from step (3) above, in warm chloroform at a temperature of at least about 45 C. up to boiling. When all the Wax is dissolved, allow the resulting warm solution to cool slowly. The first crystals, identified as CMP (cell maturation principle) which form and float on the surface of the cooling chloroform, e.g., after cooling 3-5 C. or at about 3943 C., are removed and treated as described below. With further cooling, most of anthogen Ill crystallizes as the temperature reaches 35 C. and drops to 30 C. Anthogen III, which is recovered by removing the floating crystals or by filtering or the like, is the final developer, i.e., it accelerates flowering both quantitatively and qualitatively in that it speeds up the formation of more, better and larger flowers. In the above extraction steps (2) and (3), various amounts of solvent can be employed with opti- ICC mum amounts being readily ascertainable by preliminary tests. In step (4) excess solvent is preferably avoided, the amount of warm solvent employed being about that required to readily dissolve all of the wax residue at the temperature of the solvent.

The following example will serve to illustrate the invention.

Example (1) About 1 part of commercial carnauba wax is first extracted at room temperature with about 10-12 parts (w./v.) of a refined hydrocarbon solvent, such as Skelly- C. Anthogen l is in this extract and is recovered by evaporation of the solvent. As noted above, anthogen I can be used to induce flowering in flowering plants.

(2) The wax residue from step (1) above is next extracted at room temperature with chloroform in a ratio (w./v.) of about 1 part of wax to about 16-12 parts of chloroform. Anthogen ii is in this chloroform extract and is recovered by evaporation of the solvent. As noted above, anthogen H initiates flowering in flowering plants.

(3) The wax residue from step (2) above is next dissolved in warm chloroform, e.g., 45-55 C, suflicient heat being used to dissolve all of the Wax. The use of about 10-12 parts of chloroform to about 1 part of Wax residue is adequate, e.g., l0-12 liters of Cl-lCl to 1 kilogram of wax. When the wax is dissolved, the solution is allowed to cool and as the temperature drops 35 C., e.g., from 45 to 39 (2., CM? crystals form and are separated from the solution. As the temperature of the solution continues to drop crystals continue to form and a neutral cut is removed at about 39 to 35 C. Between 353() C. another crop of crystals forms. This latter crystalline material contains anthogen ill and is removed or recovered from the solution by filtering or equivalent means. As noted above, anthogen Ill, accelerates flowering both quantitatively and qualitatively.

The anthogens are Water-insoluble complex compounds which appear to be related chemically. They appear to be lipid in character in that they are oil-soluble.

The CMP crystals obtained as above, consist essentially of potentially active anti-auxin material. The CM? crystals, for example, can be converted to a highly active anti-auxin product by redissolving the CM? crystals in hot chloroform and heating the resulting solution until the chloroform is evaporated. The residue obtained is free from auxin-like and anthogen materials and can be used to mature proliferating and callus tissues or to combat auxin activity as described in our prior patent, supra.

It will be appreciated that various fat solubilizing organic solvents and fractionating means can be employed to recover the anthogens, tree from auxin material. The anthogens, for example, are removed by hot Skelly-C at about 76 C. However, as the compositions employed in the present invention to accelerate blossoming are made up of mixtures of the anthogens, it is only necessary that the compositions consist essentially of (l) the fraction of carnauba wax that is soluble at room temperature in a refined hydrocarbon solvent, i.e., anthogen I, (2) the fraction of carnauba wax that is soluble at room temperature in chloroform, i.e., anthogen II, and (3) the fraction of carnauba wax that is soluble in warm chloroform above 35 C. but insoluble in chloroform at temperatures between 36-35" C. This composition includes each of the desired blossom generating materials.

In practice, it is preferred to mix about 1 part by volume of the mixed anthogens, as recovered from carnauba wax, with 10 parts by volume of water and about 0.5 part by volume of a standard wax suspending or emulsifying agent, e.g., a non-ionic surfactant, warm the resulting mixture until the anthogens melt, agitate vigorously as the mixture cools and continue the agitation until crystallization of the anthogens is complete. The resulting homogeneous concentrate in which the anthogens are present as dispersed microcrystals in colloidal form, is then'diluted with Water to provide a final composition containing 300 -500 ppm. of the mixed anthogens. To generate blossoming in plants, otherwise in condition to flower, spraying of the diluted aqueous composition on the tips of the plants down to the mature leaves is preferred. In addition to the socalled decorative or fioweringplants, it will be apparent to those skilled in the art that the compositions of the present invention can also be used to assist flowering, fruiting and seed production in various flowering agricultural crops.

While it is generally preferred in most cases to use a composition containing each anthogen, i.e., anthogens I, II and III, a composition containing only anthogen II and anthogen III, has been found to have utility in this field. A composition of this latter type contains the fraction of carnauba or equivalent wax which is soluble at room temperature in chloroform and the fraction of the wax which is soluble in hot chloroform above 35 C. but insoluble in chloroform between 3035 C.

It is claimed:

1. A composition characterized by its ability to generate blossoming in flowering plants, consisting essentially of (1) the fraction of carnauba wax that is soluble at a temperature of from about -22 C. in a refined hydrocarbon solvent consisting predominantly of n-heptan'e and having a boiling point of from 90-100 C., (2) the fraction of carnauba wax residue of (1) that is soluble at a temperature of from about 2022 C. in chloroform and (3) the fraction of carnauba Wax residue of (2) that is soluble in warm chloroform at temperatures above 35 C. and insoluble in chloroform at temperatures between 30-35 C.

2. In the process of preparing a composition in accordance with claim 1 consisting essentially of anthogens I, II and Ill and characterized by its ability to generate blossoming in flowering plants, the steps which comprise (1) extracting carnauba wax with a refined hydrocarbon solvent consisting predominantly of n-heptane and having 'a boiling point of from 90-100 C. at a temperature of from about 20-22 C., recovering anthogen I by removing the solvent from the resulting extract, (2) extracting t the wax residue from step (1) with chloroform at a ternperature of from about 2022 C., recovering anthogen II by removing the solvent from the resulting extract, (3) dissolving the wax residue from step (2) in hot chloroform at a temperature 'of about 55 C., cooling the resulting solution, removing crystals which form in the solution as the solution cools down to about 35 C., continue cooling the solution, and removing the crystals of anthogen III which form in the solution as the solution cools down from about 35 C. to about 30 C.

3. The process of preparing an anti-auxin composition in accordance with claim 2, which consists in removing the first crop of crystals which form at 45 to 39 C., in the cooling chloroform in step (3) of the process of claim 2, dissolving the resulting crystals in Warm chloroform, and recovering the crystalline material as an active residue by heating the resulting solution until all of the solvent is removed. 7 i

4. A composition characterized by its ability to accelerate blossoming in flowering plants containing as essential ingredients, the fraction of carnauba wax which is soluble at 'a temperature of from about 20-22 C. in chloroform and the'fraction of carnauba wax which is soluble in hot chloroform above 35 C. and insoluble in chloroform between about 30-35 C., said fractions corresponding to the fractions obtained in steps (2) and (3) of'claim 2.

5. The process of claim 3, where the crystals which form at 39 to 35 C. are removed from the cooling chloroform.

References Cited by the Examiner UNITED STATES PATENTS 5/60 Roberts et al. 7l-2.7

OTHER REFERENCES Roberts, Science, vol. 117 (1953), pages 456 and 457.

IRVING MARCUS, Primary Examiner.

MAURICE A. BRINDISI, NICHOLAS S. RIZZO,

WALTER A. MODANCE, Examiners. 

1. A COMPOSITION CHARACTERIZED BY ITS ABILITY TO GENERATE BLOSSOMING IN FLOWERING PLANTS, CONSISTING ESSENTIALLY OF (1) THE FRACTION OF CARNAUBA WAX THAT IS SOLUBLE AT A TEMPERATURE OF FROM ABOUT 20-22*C. IN A REFINED HYDROCARBON SOLVENT CONSISTING PREDOMINANTLY OF N-HEPTANE AND HAVING A BOILING POINT OF FROM 90-100*C., (2) THE FRACTION OF CARNAUBA WAX RESIDUE OF (1) THAT IS SOLUBLE AT A TEMPERATURE OF FROM ABOUT 20-22*C. IN CHLOROFORM AND (3) THE FRACTION OF CARNAUBA WAX RESIDUE OF (2) THAT IS SOLUBLE IN WARM CHLOROFORM AT TEMPERATURES ABOVE 35* C. AND INSOLUBLE IN CHLOROFORM AT TEMPERATURES BETWEEN 30-35*C.
 2. IN THE PROCESS OF PREPARING A COMPOSITION IN ACCORDANCE WITH CLAIM 1 CONSISTING ESSENTIALLY OF ANTHOGENS I, II AND III AND CHARACTERIZED BY ITS ABILITY TO GENERATE BLOSSOMING IN FLOWERING PLANTS, THE STEPS WHICH COMPRISE (1) EXTRACTING CARNAUBA WAX WITH A REFINED HYDROCARBON SOLVENT CONSISTING PREDOMINANTLY OF N-HEPTANE AND HAVING A BOILING POINT OF FROM 90-100*C. AT A TEMPERATURE OF FROM ABOUT 20-22*C., RECOVERING ANTHOGEN I BY REMOVING THE SOLVENT FROM THE RESULTING EXTRACT, (2) EXTRACTING THE WAX RESIDUE FROM STEP (1) WITH CHLOROFORM AT A TEMPERATURE OF FROM ABOUT 20-22*C., RECOVERING ANTHOGEN II BY REMOVING THE SOLVENT FROM THE RESULTING EXTRACT, (3) DISSOLVING THE WAX RESIDUE FROM STEP (2) IN HOT CHLOROFORM AT A TEMPERATURE OF ABOUT 45-55*C., COOLING THE RESULTING SOLUTION, REMOVING CRYSTALS WHICH FORM IN THE SOLUTION AS THE SOLUTION COOLS DOWN TO ABOUT 35*C., CONTINUE COOLING THE SOLUTION, AND REMOVING THE CRYSTALS OF ANTHOGEN III WHICH FORM IN THE SOLUTION AS THE SOLUTION COOLS DOWN FROM ABOUT 35*C. TO ABOUT 30*C. 